Sulpizi M, Laio A, VandeVondele J, Cattaneo A, Rothlisberger U, Carloni P
SISSA, International School for Advanced Studies, Trieste, Italy.
Proteins. 2003 Aug 1;52(2):212-24. doi: 10.1002/prot.10275.
Caspases are fundamental targets for pharmaceutical interventions in a variety of diseases involving disregulated apoptosis. Here, we present a quantum mechanics/molecular mechanics Car-Parrinello study of key steps of the enzymatic reaction for a representative member of this family, caspase-3. The hydrolysis of the acyl-enzyme complex is described at the density functional (BLYP) level of theory while the protein frame and solvent are treated using the GROMOS96 force field. These calculations show that the attack of the hydrolytic water molecule implies an activation free energy of ca. DeltaF(A) approximately equal 19 +/- 4 kcal/mol in good agreement with experimental data and leads to a previously unrecognized gem-diol intermediate that can readily (DeltaF(A) approximately equal 5 +/- 3 kcal/mol) evolve to the enzyme products. Our findings assist in elucidating the striking difference in catalytic activity between caspases and other structurally well-characterized cysteine proteases (papains and cathepsins) and may help design novel transition-state analog inhibitors.
半胱天冬酶是各种涉及细胞凋亡失调疾病的药物干预的基本靶点。在此，我们展示了对该家族代表性成员半胱天冬酶-3酶促反应关键步骤的量子力学/分子力学Car-Parrinello研究。酰基酶复合物的水解在密度泛函（BLYP）理论水平上进行描述，而蛋白质框架和溶剂则使用GROMOS96力场处理。这些计算表明，水解水分子的攻击意味着约19±4千卡/摩尔的活化自由能，与实验数据吻合良好，并导致一种先前未被识别的偕二醇中间体，该中间体可以很容易地（活化自由能约为5±3千卡/摩尔）演变为酶产物。我们的研究结果有助于阐明半胱天冬酶与其他结构特征明确的半胱氨酸蛋白酶（木瓜蛋白酶和组织蛋白酶）之间催化活性的显著差异，并可能有助于设计新型过渡态类似物抑制剂。
